Mechanisms of size exclusion chromatography

Size exclusion chromatography separates molecules according to their size in solution. As a sample passes through the column, molecules which are too large to penetrate the pores of the packing are excluded and remain in the interparticle volume, Vq (this is also called the void or interstitial volume and is not to be confused with the solvent elution time Iq normally encountered in other forms of liquid chromatography). These molecules are eluted first from the column at the point of total exclusion. Small molecules which can permeate all the pores elute at the solvent front or total solvent volume F. This is the point of total permeation. Molecules of intermediate size will penetrate 

This curve is constructed by measuring the peak elution volumes (Fg) of a series of monodisperse polymer standards with known peak molecular masses. The elution volumes of these materials decrease with increase in the logarithm of the molecular mass [15]. 

Polymers with differing chemical structures will usually have a different coil volume in solution and produce a displaced calibration curve. Various procedures have been proposed to overcome this problem and to produce a Universal calibration curve. These include the g factor method [16], the use of hydrodynamic volume [17], and unperturbed dimensions [18]. The use of broad-molecular-mass distribution polymers as calibration standards has also been employed. These procedures are discussed more fully in Chapter 3. They are only important if accurate relative molecular masses are required if the technique is being used simply for qualitative purposes, these procedures are not important. 

This approximates to the capacity ratio normally encountered in interactive chromatographic processes where the numerator in eqn (1.14) is equivalent to the stationary phase and the denominator to the mobile phase. In SEC, is always between zero and one. For the largest molecules, k = 0 (i.e. = Vq  

As all the components of a sample elute between Vq and (Vq -f V-X and in order to maximize the separation, it is important to use columns with a large pore volume and to minimize the void volume. The former is fixed by the nature of the column packing material, but the chromatographer has some 

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Potschka, M., Mechanism of size-exclusion chromatography. I. Role of convection and obstructed diffusion in size-exclusion chromatography, /. Chromatogr., 648, 41, 1993. 

Figure 13.28 Mechanism of size exclusion chromatography (a) size selectivity as a function of pore and analyte sizes and (b) Separation process on a Gel-Filtration Column. (Adapted with permission from Phenomenex, Inc.)...

Fig.l. Calibration curve for the mechanism of size-exclusion chromatography under ideal conditions ... 

Since the inception of size exclusion chromatography, many models have been considered in an attempt to describe the retention of macromolecules. Most of these models treated the pores as having various well-defined shapes, and examined the effects of purely physical exclusion mechanisms. In practice, these models gave good agreement with observed retention behaviour. 

A number of analytical techniques such as FTIR spectroscopy,65-66 13C NMR,67,68 solid-state 13 C NMR,69 GPC or size exclusion chromatography (SEC),67-72 HPLC,73 mass spectrometric analysis,74 differential scanning calorimetry (DSC),67 75 76 and dynamic mechanical analysis (DMA)77 78 have been utilized to characterize resole syntheses and crosslinking reactions. Packed-column supercritical fluid chromatography with a negative-ion atmospheric pressure chemical ionization mass spectrometric detector has also been used to separate and characterize resoles resins.79 This section provides some examples of how these techniques are used in practical applications. 

The two techniques differ in that HDC employs a nonporous stationary phase. Separation is affected as a result of particles of different size sampling different velocities in the interstitial spaces. Size exclusion chromatography is accomplished by superimposing a steric selection mechanism which results from the use of a porous bed. The pore sizes may vary over a wide range and the separation occurs as a result of essentially the same processes present in the gel permeation chromatography of macromolecules. 

Watson and Kenney [62] describe the use of high-performance size-exclusion chromatography to examine the aggregation of interferon-y and interleukin-2 after storage at elevated temperature, after mechanical agitation, and following rapid freeze-thaw. An excellent review on SEC can be found in Ref. 63. 

We report here the results of our recent studies of poly(alkyl/arylphosphazenes) with particular emphasis on the following areas (1) the overall scope of, and recent improvements in, the condensation polymerization method (2) the characterization of a representative series of these polymers by dilute solution techniques (viscosity, membrane osmometry, light scattering, and size exclusion chromatography), thermal analysis (TGA and DSC), NMR spectroscopy, and X-ray diffraction (3) the preparation and preliminary thermolysis reactions of new, functionalized phosphoranimine monomers and (4) the mechanism of the polymerization reaction. 

The data prove that the retention order of anthocyanins deviates from each other in HPLC and TLC suggesting the involvement of a different retention mechanism. It was stated that the preseparation of anthocyanins by size-exclusion chromatography is a prerequisite of the successful preparative separation by RP-HPLC [244],... 

There are four different mechanisms of separation utilized in HPLC adsorption, partition, ion-exchange, and size exclusion chromatography. 

In the first section, the mechanisms involved in size exclusion chromatography are discussed this is an area where additional understanding and clarification still are needed. Data treatment with respect to statistical reliability of the data along with corrections for instrumental broadening is still a valid concern. Instrumental advances in the automation of multiple detectors and the developm.ent of a pressure-programmed, controlled-flow supercritical fluid chromatograph are presented. 

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